Lipid-lowering quinazoline derivative

ABSTRACT

A novel carbonyl-substituted quinazoline, preferably 4-(3&#39;-bromobenzoyl)-6,7-dimethoxyquinazoline (WHI-P164), and methods for lowering blood cholesterol, including reducing total cholesterol and LDL-cholesterol levels by administration of the carbonyl quinazolines and compositions thereof.

This application is a continuation of application Ser. No. 09/126,940,filed Jul. 30, 1998, now U.S. Pat. No. 6,172,071, application(s) areincorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to novel quinazoline derivatives, for example4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline), and methods of use. Inparticular, the compounds of the invention, when administered to asubject, are effective in reducing blood cholesterol levels in thesubject.

BACKGROUND OF THE INVENTION

Atherosclerosis and ischemic heart disease remain the major cause ofdeath of Americans. Elevated serum cholesterol levels present a majorrisk factor for atherosclerosis and related complications includingmyocardial infarction, heart failure, and cerebral stroke. Interventionstudies performed in middle-aged men demonstrated a marked reduction inthe incidence of cardiovascular events after the lowering of elevatedtotal and low-density lipoprotein cholesterol (LDL-C) levels. TheCholesterol and Recurrent Events (CARE) trial and the ScandinavianSimvastatin Survival Study (4S)(1994, Lancet 344:1383-1389) have furthershown that both women and elderly patients with prior history ofischemic heart disease benefit from cholesterol lowering therapy.(Miettinen et al., 1988, Arch. Intern. Med. 148:36-69; Sacks et al.,1996, New Eng. J Med. 335:1001-1009)

The most effective cholesterol lowering drugs are statins, which lowercholesterol levels by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA) reductase, an enzyme which catalyzes the limiting step incholesterol biosynthesis (Goldstein et.al., 1984, J. Lipid Res. 25,1450-1461). Compared to treatment regimens with other lipid loweringagents, such as the bile acid sequestrants (colestipol andcholestyramine), nicotinic acid, fibric acid (gemfibrozil andclofibrate), probucol, and experimental ACAT inhibitors, statin therapyhas been found to bear several favorable features. Statins achieveextensive lowering of LDL-C, leading to an overall reduction inmortality, and are cost effective due to substantial reduction ofhospital admissions and rates of coronary intervention. Statins alsoachieve better compliance than other treatments, as a result of theironce-daily administration and few side effects. Combination of statinswith other agents is considered necessary for patients with severe,complex or refractory lipid disorders. Furthermore, large clinicaltrials have suggested regression of atherosclerotic lesions byaggressive lipid lowering therapy (Schell and Myers, 1997, Prog. onCardiovascular Diseases 39:483-496). To complement the statins andachieve successful reduction of cholesterol in statin-resistantsubjects, identification of new lipid lowering agents with a differentmechanism of action than statins remains a major focal point incontemporary atherosclerosis research. Since the HMG CoA inhibitors areineffective in the mouse, this animal provides a useful model forscreening novel agents capable of lowering cholesterol levels by adifferent mechanism of action than statins.

There is a need for new lipid lowering agents that are effective tolower total cholesterol and/or LDL-C, for the treatment of highcholesterol and other lipid disorders including those which are severe,complex, and/or refractory to current treatments.

SUMMARY OF THE INVENTION

Quinazoline compounds having a carbonyl substitution (carbonyl-Q) asdescribed below and exemplified by4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline (WHI-P164) have now beenidentified as a new class of potent cholesterol lowering agents. Asshown in the Examples below, administration of the WHI-P164 reducestotal cholesterol levels in hypercholesterolemic C57B1/6 mice on a highcalorie diet by 23% and LDL-C by 45%. WHI-P164 also reduced totalcholesterol levels and β-VLDL/LDL-cholesterol levels ofhypercholesterolemic apolipoprotein E deficient mice (apo e^(−/−)) by41% and 63%, respectively.

The present invention provides potent cholesterol-lowering agents,quinazoline compounds having a carbonyl group, (carbonyl-Q). Anexemplary compound of the invention is 4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline).

The novel cholesterol lowering agents of the invention may be formulatedby means known in the art for delivery to targeted areas of the body,including blood and/or gut, for example, by choice of carrier, mode ofadministration, or by conjugating carbonyl-Q with a specific targetingmoiety, such as an antibody or ligand which binds a specific antigen orligand receptor in the target tissue. Formulations useful fortherapeutic reduction of cholesterol include injectable compositions,oral compositions, depot formulations, and the like containing aneffective cholesterol-lowering amount of a carbonyl-Q compound of theinvention, such as 4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline.

In the methods of the invention, carbonyl-Q cholesterol lowering agentssuch as 4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline are administered toa subject in order to modulate lipids in the blood, and particularly tolower blood cholesterol.

The above summary of the present invention is not intended to describeeach disclosed embodiment or every implementation of the presentinvention. The examples and the detailed description which follow moreparticularly exemplify these embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the reduction of VLDL-C by WHI-P164 treatmentin the C57B1/6 hypercholesterolemia model. Cholesterol profiles ofC57B1/6 mice fed with a cocoa butter diet and 0 mg/kg/day (opencircles); 1.6 mg/kg/day (open squares); or 16 mg/kg/day (closed circles)(n=4) are shown.

FIG. 2 is a graph showing reduction of VLDL-C by WHI-P164 treatment inapo-e^(−/−) mice fed on rodent chow and treated with WHI-P164 at 8mg/kg/day for 7 days (closed circles, n=8) or with vehicle alone (opencircles, n=4).

FIG. 3 is a graph showing reduction of VLDL-C by WHI-P164 treatment inapo-e^(−/−) mice fed with a high fat, high cholesterol diet. Cholesterolprofiles of apo-e^(−/−) mice fed a Western diet and treated withWHI-P164 at 40 mg/kg/day for 7 days (closed circles, n=2) or withvehicle alone (open circles, n=3) are shown.

FIGS. 4A-4D are photographs showing reduction of hepatic triglyceridesynthesis by WHI-P164 treatement. Livers of apo-e^(−/−) mice fed aWestern diet and treated with vehicle alone (FIGS. 4A and 4C) or with 1mg/day of WHI-P164 for one week (FIGS. 4B and 4D) were analyzed usingOil Red O staining (FIGS. 4A and 4B) and Haemotoxylin and Eosin tissuesection staining (FIGS. 4C and 4D).

FIG. 5 is a graph showing inhibition of postprandial triglycerideaccumulation by WHI-P164 treatment. C57B1/6 mice were treated withWHI-P164 at 40 mg/kg/day for 7 days, and fasted 36 hours with continuingtreatment. Food was readministered at 0, 1, 3, 6, and 9 hours. WHI-P164treated mice (closed circles) exhibited much reduced postprandialtriglyceride accumulation in comparison to vehicle treated mice (opencircles).

FIGS. 6A-D demonstrate WHI-P164 induced regression of pre-existingatherosclerotic lesions in hypercholesterolemic apo-e^(−/−) mice. Aortasfrom untreated (FIG. 6A), vehicle treated (FIG. 6B), and WHI-P164treated (FIG. 6C) animals were stained with Sudan IV to show the fattystreaks. Aortas of untreated control mice were obtained at 7 months toreflect the pretreatment condition of the test groups. Aortas ofvehichle treated or WHI-P164 treated test mice were obtained at 8 monthsfollowing a one month treatment program. Statistical comparisonsindicate **p=0.002 for untreated controls versus WHI-P164 treated testmice and p=0.007 for vehicle controls versus WHI-P164 treated mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention includes a cholesterol lowering compound comprising acarbonyl substituted quinazoline (carbonyl-Q) exemplified by4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline (WHI-P164) and a method forlowering blood cholesterol by administration of a carbonyl-Q.

Cholesterol-Lowering Compounds of the Invention

The cholesterol-lowering compounds of the invention includecarbonyl-substituted quinazolines (carbonyl-Q) having the followinggeneral structural formula:

where X is an alkyl (straight chain, branched, or cyclic) or is anaromatic ring structure, such as phenyl, napthyl, and the like. X may besubstituted, for example, with a halogen, OH, SH, alkyl, alkoxy,acyloxy, NH₂, and the like. Carbonyl-Q may be substituted, for example,carbon or nitrogen atoms at positions 2, 5, 6, or 8 may have boundthereto halo, H, OH, alkyl, alkoxy, acyloxy, and the like groups.

An exemplary carbonyl-Q compound of the invention is4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline (WHI-P164), having thefollowing structure:

The cholesterol-lowering compounds of the invention are formulated intocompositions for administration, prefereably including a carrier whichassists in the administration of the compound for cholesterol-loweringeffects. For example, the composition may include a carrier which aidsin suspending or solubilizing the compound of the invention or toimprove the flavor or texture of the composition in foodstuffs orbeverages for oral delivery to the gut. Alternatively, a compositioncontaining the compound of the invention may include an isotonic carrierto facilitate delivery by injection, agents to prolong the half-life ofthe compound, and the like.

Multiple delivery systems are known for delivery of compounds to thebloodstream and gut of an animal. Preferably, the compound of theinvention is administered to the gut, most preferably by oral deliveryin a foodstuff composition, such as a dietary supplement or a staplefoodstuff supplemented with the composition of the invention.

Mode of Administration

The cholesterol-lowering compounds of the invention can be formulated aspharmaceutical compositions, nutritional supplements, or as additives infoodstuffs and administered to a mammalian host, including a human in avariety of forms adapted for administration of a quinazoline compound.Preferred administration routes include intravenous, intramuscular, andsubcutaneous injection. Most preferred is oral administration.

Solutions or suspensions of the cholesterol-lowering composition can beprepared in water, isotonic saline (PBS) and may preferably be mixedwith a non-toxic surfactant. Dispersions may also be prepared inglycerol, vegetable oils, and the like. Under ordinary conditions ofstorage and use, these preparations may contain a preservative toprevent the growth of microorganisms.

The dose of the composition to be administered can vary widely inaccordance with the age, size, and condition of the subject to betreated. Useful dosages of the composition are those which will deliverabout 0.1 to about 500 mg/kg body weight/day, and preferably deliverabout 0.5 to about 10 mg/kg body weight/day. The amount of the compoundneeded in the composition can vary with the mode of administration,e.g., by injection or by oral administration, to account for variance inthe metabolism of the compound and of the composition.

While the invention is amenable to various modifications and alternativeforms, specific embodiments of the invention are shown by way of theExamples and will be described in detail. It should be understood,however, that the intention is not to limit the invention to theparticular embodiments described. On the contrary, the intention is tocover all modifications, equivalents, and alternatives falling withinthe spirit and scope of the invention.

EXAMPLES

The invention may be better understood by reference to the followingExamples, that are not intended to limit the invention in any way.

Example 1 Animal Models of Hypercholesterolemia and Analytical Methods

Three- to four-week old C57B1/6 male mice (Taconic, Germantown, N.Y.,USA) were kept in micro-isolator cages on a 12-hour day/night cycle andfed the Paigen's cocoa butter diet (15.8% fat, 1.25% cholesterol, and0.5% sodium cholate) (Harlan Teklad, Madison, Wis., USA) for two weeksprior to initiation of lipid lowering therapy. Paigen's diet isdescribed in Nishina et.al., 1993, J. Lipid Res. 34: 1413-1422.

Three- to four-week old apolipoprotein E deficient (apo E^(−/−)) micewere the progeny of breeding pairs of apo E knockout (apo EKO) miceobtained from Jackson Labs. (Bar Harbor, Me., USA). Mice were fed eitherregular rodent chow (5% fat and 0% cholesterol) (Teklad-LM 485, HarlanTeklad, Madison, Wis., USA), the Paigen's cocoa butter diet, or theWestern diet (21.22% fat and 0.2% cholesterol) (Harlan Teklad, Madison,Wis., USA) for two weeks prior to treatment.

Animals were treated with test compounds by intraperitoneal injectiondaily for 7 days. A range of doses was administered. Control animalsreceived vehicle alone. The effect of quinazoline derivatives on totalcholesterol, LDL, HDL, liver enzymes, and glucose was analyzed. The micetissues were also examined histologically.

Lipid determinations:

Serum total cholesterol was measured enzymatically using the cholesterolkit from Sigma Chemical Co. (St. Louis, Mo., USA). For liver tissuelipid measurements, lipids were extracted from homogenized hepatictissues using the method of Bligh and Dyer (Bligh, et al., Can. JBiochem. Phys. 1959, 37:911-917). One gram of liver tissue washomogenized in 1 ml of distilled water and extracted with 4 ml ofchloroform/methanol (1:1, v/v). The chloroform phase containing thelipids was evaporated under a stream of nitrogen, the lipids dissolvedin 100 μL of isopropanol, and subjected to cholesterol determination asdescribed above. Triglyceride (TG) contents were determined using the TGkit from Sigma Chemical Co.

Serum cholesterol profiles were examined by Fast Protein LiquidChromatography (FPLC) using 100 μl of serum per run. The serum waspassed over two Superose™ 6 HR 10/30 connected in tandem equilibrated inphosphate buffered saline (PBS), using the FPLC system from PharmaciaBiotech (Piscataway, N.J., USA) consisting of a controller LCC-501 plusconnected to a UV detector (UV-MII), two P500 pumps, and a Frac-100fraction collector. The FPLC was remotely controlled and operated by theFPLC director program operated on an IBM computer. The system wasoperated at a flow rate of 0.5 ml/minute and fractions were collected at0.5 ml/fraction. Cholesterol concentration of each fraction wasdetermined as described above and plotted against the fraction numbersto obtain the serum cholesterol profile.

To determine the serum concentrations of LDL/β-VLDL and HDL, the sums ofthe LDL/β-VLDL peak (fractions 29-35) and the HDL peak (fractions 53-63)were divided by 0.1. To obtain the IC₅₀ and the minimum β-VLDL and HDLconcentrations, the concentrations of β-VLDL and HDL were plottedagainst the dosage. Best fitted exponential decay curves were obtainedusing the equation: Concentration of HDL or LDL/β-VLDL=Aexp(−BX)+E,where A+E equals the concentration of HDL or LDL/β-VLDL at the dosage of0 μg/day and E equals the mathematical minimum concentration of HDL orβ-VLDL, and B equals 0.69/IC₅₀. Best fitted curves were obtained usingthe Graphpad Inplot program, Graphpad Software, San Diego, Calif., USA.

Determination of In Vivo Hepatic VLDL-TG Production Using Triton WR1339

C57B1/6 and apoE-deficient mice were treated with intraperitonealinjections of 50 μl vehicle or 40 mg/kg WHI-P164 in 50 μl vehicle for 7consecutive days, followed by a 36 hour fast to shut down intestinalchylomicron synthesis. Subsequently, mice were injected intravenouslywith 500 mg/kg Triton WR1339 (Sigma Chemical Co.) in 0.9% NaCl tocompletely inhibit their plasma VLDL clearance, as previously reported(Pasternali et al., 1996, Ann. Intern. Med. 125:529-540; Aalto-Setala etal., 1992, J. Clin. Invest. 90:1889-1900). Blood samples were taken at 0and 4 hours after Triton WR1339 injection and plasma TG levels weremeasured enzymatically using a commercially available enzymatic kit(Sigma Chemical Co.). Alternatively, food was returned to the mice aftera 36 hour fast and blood samples were collected at 0, 1, 3, 6, and 9hours after returning the food. Accumulation of chylomicron-TG wasdetermined as accumulation of plasma TG.

Blood Chemistry

Serum levels alanine aminotransferase (ALT), alkaline phosphatase (ALP),lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine,creatinine phosphokinase (CPK), and glucose were determined by using aSynchron CX5 Clinical System (Beckman Instruments, Inc., Fullerton,Calif.) following manufacturer's instructions. For the glucose tolerancetest, blood glucose levels were determined using the portable One TouchProfile glucose meter (Lifescan, Milpitas, Calif., USA).

Histopathology

At the end of the experiment, the animals were sacrificed and theharvested tissues were fixed in 10% phosphate buffered formalinovernight, embedded, sectioned, and stained by hematoxylin and eosin(H&E). The stained sections were examined for pathological changes. ForOil Red O staining, the liver was excised and snap frozen in liquidnitrogen. Five micrometer thick sections were obtained using theLeicaCM1800 cryostat (Heerbrugg, Switzerland), stained in 0.5% Oil Red Oin propylene glycol for 1 hour, destained in 85% propylene glycol for 1minute, rinsed twice in distilled water, counterstained in Mayer'shematoxylin for a few seconds, rinsed twice in distilled water, andmounted in Crystalmount (Biomedia Corp., Foster City, Calif.). The OilRed O stainable material at 400× magnification was quantitated using theImagePro plus program (Media Cybernetics, Silver Spring, Md., USA) inconjunction with a 3CCD camera (DAGE-MTI Inc., Michigan City, USA).

Example 2 Chemical synthesis and Characterization of QuinazolineDerivatives

The common synthetic intermediate 4-chloro-6,7-dimethoxyquinazoline 5used for synthesizing all the tested compounds by following literatureprocedures (Scheme 1). 4,5-dimethoxy-2-nitrobenzoic acid 1 was treatedwith thionyl chloride, and then reacted with ammonia to give4,5-dimethoxy-2-nitrobenzamide 2 (Nomoto et al., 1990, Chem. Pharm. Bull38: 1591-1595). The nitro group in compound 2 was reduced with sodiumborohydride in the presence of copper sulfate (31) to give4,5-dimethoxy-2-aminobenzarmide 3, which was cyclized by refluxing withformic acid to give 6,7-dimethoxyquinazoline-4(3H)-one 4. Thequinazolinone 4 was refluxed with phosphorus oxytrichloride to providethe key starting material 5 with good yield.

The lead compound 4-(3′-bromobenzoyl)-6,7-dimethoxyquinazoline WHI-P164was synthesized by reacting 4-chloro-6,7-dimethoxyquinazoline 5 with thecommercially available 3-bromobenzaldehyde in the presence of1,3-dimethylimidazolium iodide and sodium hydride in refluxing dioxanefor 4 hours (Scheme 2) (32,33). The remaining 6,7-dimethoxyquinazolinederivatives were synthesized by condensing4-chloro-6,7-dimethoxyquinazoline 5 with the corresponding substitutedanilines as shown in Scheme 3.

Synthetic Procedures and characterization data: All chemicals werepurchased from Aldrich (Milwaukee, Wis.) or Sigma (St. Louis, Mo.) andwere used without further purification. Except where distinguished, eachreaction vessel was secured with a rubber septa, and the reaction wasperformed under nitrogen atmosphere. ¹H and ¹³C NMR spectra wereobtained on a Varian Mercury 300 instrument at ambient temperature inDMSO-d₆. Melting points were determined using a Fisher-Johns meltingpoint apparatus and are uncorrected. FT-IR spectra were recorded on aNicolet Protege 460 spectrometer. GC/MS was obtained on a HP 6890 GCSystem equipped with a HP 5973 Mass Selective Detecter. TLC wasperformed on a precoated silica gel plate (Silica Gel KGF; Whitman Inc).Silica gel (200-400 mesh, Whitman Inc.) was used for all columnchromatography separation.

4,5-Dimethoxy-2-nitrobenzamide 2. A suspension of4,5-dimethoxy-2-nitrobenzoic acid 1 (2 g; 8.8 mmol) in SOCl₂ (10 mL) wasstirred under reflux for 50 minutes. After cooling, the reaction mixturewas poured into a mixture of concentrated NH₄OH (50 mL) and ice (30 g).The precipitate were collected by filtration, washed with water, anddried to give 1.85 g crude crystals. After recrystallization from DMF,1.76 g pure product was obtained (88.5%). ¹H NMR(DMSO-d₆): δ 7.60(s, 2H,—NH₂), 7.57(s, 1H, 6-H), 7.12(s, 1H, 3-H), 3.90, 3.87(s, s, 6H, —OCH₃);IR(Kr) υ_(max): 3454, 2840, 1670, 1512, 1274, 1227 cm⁻¹; GC/MS m/z226(M⁺, 10.0), 178(98.5), 163(100.0), 135(51.0).

6,7-Dimethoxyquinazoline-4(3H)-one 4. NaBH₄ (400 mg) was added withstirring over 4 hours to a solution of 4,5-dimethoxy-2-nitrobenzamide 2(1.58 g; 7 mmol) in MeOH containing catalytic amount of CuSO₄. Thereaction mixture was poured into ice-water (200 mL) with stirring togive 4,5-dimethoxy-2-aminobenzamide 3 which was directly refluxed withHCOOH (20 mL) for 5 hours. After removal of solvent, the residue wasrecrystallized from DMF to give pure crystals 4 (1.18 g; 81.5%). m.p.295.0-297.0° C.; ¹H NMR (DMSO-d₆): δ 12.03 (br, s, 1H, —NH), 7.99 (s,1H, 2-H), 7.42 (s, 1H, 5-H), 7.11 (s, 1H, 8-H), 3.88, 3.85 (s, s, 6H,—OCH₃); IR(KBr)υ_(max): 3015, 2840, 1648, 1504, 1261, 1070 cm⁻¹; GC/MSm/z 206(M⁺, 100), 191(M⁺ —CH₃, 31.5), 163(16.7), 120(15.2).

4-Chloro-6,7-dimethoxyquinazoline 5. A suspension of6,7-dimethoxyquinazoline-4(3H)-one 4 (12.36 g, 60 mmol) in POCl₃ (250mL) was heated under reflux for 4 hr, when a clear solution wasobtained. The POCl₃ was removed under reduced pressure, and the residuewas dissolved in a mixture of CH₂Cl₂ and aqueous Na₂CO₃. The organiclayer was dried and the solvent removed to give4-chloro-6,7-dimethoxyquinazoline 5 (11.2 g, 83%); m.p. 259.0-263.0° C.;¹H NMR (DMSO-d₆): d 8.75(s, 1H, 2-H), 7.53(s, 1H, 5-H), 7.25(s, 1H, 8H),3.91(s, 3H, —OCH₃), 3.89(s, 3H, —OCH₃); IR (KBr) u_(max): 2963, 2834,1880, 1612, 1555, 1503, 1339, 1153, 962 cm⁻¹. GC/MS m/z 224(M⁺, 100),209(M⁺ —CH₃, 9.4), 189(19.39), (69(10.55).

4-(3′-Bromobenzoyl)-6,7-dimethoxyquinazoline WHI-P164. Sodium hydride(108 mg; 4.5 mmol) was added to a stirred solution of4-chloro-6,7-dimethoxyquinazoline 5 (896 mg; 4 mmol),3-bromobenzaldehyde (832 mg; 4.5 mmol) and 1,3-dimethylimidazoliumiodide (336 mg; 1.5 mmol) in dioxane (30 mL) and the mixture wasrefluxed for 4 hours. The reaction mixture was cooled to roomtemperature, poured into ice-water, and precipitate was collected. Theyield was 81.2% (1.05 g). m.p. 258.0-263.0° C.; ¹H NMR (DMSO-d₆): d 9.25(s, 1H, 2-H), 8.14 (s, 1H, 5-H), 7.92-7.43 (m, 4H, 2′, 4′, 5′, 6′-H),7.40 (s, 1H, 8-H), 4.11 (s, 3H, —OCH₃), 4.00 (s, 3H, —OCH₃). IR (KBr)u_(max): 3432, 1664, 1504, 1431, 1230 cm⁻¹. GC/MS m/z 374(M⁺+1, 48.96),373(M⁺, 34.93), 372(M⁺−1,47.67), 357(58.74), 343(100.00), 293(M⁺—Br,31.48), 189(26.27).

General procedures for compounds synthesized according to Scheme 3. Amixture of 4-chloro-6,7-dimethoxyquinazoline 5 (448 mg; 2 mmol) and theappropriately substituted aniline (2.5 mmol) in 20 ml of alcohol (EtOHor MeOH) was heated to reflux. Heating was continued for 4-24 hours,sufficient Et₃N was added to neutralize the solution, and the solventwas then concentrated to give crude product, which was recrystallizedfrom DMF.

4-(3′,5′-Dibromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazolineWHI-P97: yield: 72.80%; m.p. >300.0° C.; ¹H NMR (DMSO-d6): δ 9.71 (s,1H, —NH), 9.39 (s, 1H, —OH), 8.48 (s, 1H, 2-H), 8.07 (s, 2H, 2′,6′-H),7.76 (s, 1H, 5-H), 7.17 (s, 1H, 8-H), 3.94 (s, 3H, —OCH₃), 3.91 (s, 3H,—OCH₃); IR (KBr) υ_(max): 3054 (br), 3419, 2868, 1627, 1512, 1425, 1250,1155 cm-1; GC/MS m/z 456 (M⁺+1. 54.40), 455 (M⁺, 100.00), 454 (M⁺−1,78.01), 439 (M⁺ —OH, 7.96), 376 (M⁺+1-Br, 9.76), 375 (M⁺ —Br, 10.91),360 (5.23)

4-(3′-Bromo-4′-methylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P111.yield: 82.22%; m.p.225.0-228° C. ¹H NMR (DMSO-d₆): δ 10.23(s, 1H, —NH),8.62(s, 1H, 2-H), 8.06(d, 1H,J_(2′,5′)=2.1 Hz, 2′-H), 7.89(s, 1H, 5-H),7.71(dd, 1H, J_(5′,6′)=8.7 Hz, J_(2′,6′)=2.1 Hz, 6′-H), 7.37(d, 1H,J_(5′,6′)=8.7 Hz, 5′-H), 7.21(s, 1H, 8-H), 3.96(s, 3H, —OCH₃), 3.93(s,3H, —OCH₃). IR(KBr) υ_(max): 3431, 3248,2835, 1633, 1517, 1441, 1281,1155 cm⁻¹. GC/MS m/z 375 (M⁺+1 ,76.76), 374 (M⁺,100.00), 373(M⁺−1,76.91), 358 (M⁺+1-OH, 11.15), 357(1.42), 356(6.31).

4-(4′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P131. yield:84.29%; m.p. 245.0-248.0 ° C. ¹H NMR(DMSO-d₆): d 11.21(s, 1H, —NH),9.70(s, 1H, —OH), 8.74(s, 1H, 2-H), 8.22(s, 1H, 5-H), 7.40(d, 2H,J=8.9Hz, 2′,6′-H), 7.29(s, 1H, 8-H), 6.85(d, 2H, J=8.9 Hz, 3′,5′-H), 3.98(s,3H, —OCH₃), 3.97(s, 3H, —OCH₃). IR(KBr) υ_(max): 1635, 1516, 1443, 1234cm⁻¹. GC/MS m/z 298 (M⁺+1, 100.00), 297(M⁺, 26.56), 296(M⁺−1, 12.46).

4-(2′-Hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P132. yield:82.49%; m.p. 255.0-258.0° C.; ¹H NMR(DMSO-d₆): d 9.78 (s, 1H, —NH), 9.29(s, 1H, —OH), 8.33 (s, 1H, 2-H), 7.85 (s, 1H, 5-H), 7.41-6.83 (m, 4H,3′,4′,5′,6′-H), 7.16 (s, 1H, 8-H), 3.93 (s, 3H, —OCH₃), 3.92 (s, 3H,—OCH₃); IR(KBr) υ_(max): 3500 (br), 3425, 2833, 1625, 1512, 1456, 1251,1068 cm⁻¹; GC/MS m/z 298(M⁺+1,8.91), 297(M⁺, 56.64), 281(M⁺+1-OH,23.47), 280(M⁺ —OH, 100.00).

4-(3′-Bromo-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P154.yield: 89.90%; m.p. 233.0-233.5° C.; ¹H NMR(DMSO-d₆): δ 10.08(s, 1H,—NH), 9.38(s, 1H, —OH), 8.40(s, 1H, 2-H), 7.89(d, 1H, J_(2′,5′)=2.7 Hz,2′-H), 7.75(s, 1H, 5-H), 7.55(dd, 1H, J_(5′,6′)=9.0 Hz, J_(2′,6′)=2.7Hz, 6′-H), 7.14(s, 1H, 8-H), 6.97(d, 1H, J_(5′,6′)=9.0 Hz, 5′-H),3.92(s, 3H, —OCH_(ER)), 3.90(s, 3H, —OCH₃); IR (KBr) υ_(max): 3431(br),2841, 1624, 1498, 1423, 1244 cm⁻¹; GC/MS m/z 378(M⁺+2, 90.68),377(M⁺+1,37.49), 376(M⁺, 100.00), 360(M⁺3.63), 298(18.86), 282 (6.65).

4-(3′-Chloro-4′-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline WHI-P197.yield 84.14%; m.p. 245.0° C.(dec); ¹H NMR(DMSO-d₆): δ 10.00 (s, 1H,—NH), 9.37 (s, 1H, —OH), 8.41 (s, 1H, 2-H), 7.78 (s, 1H, 5-H), 7.49 (d,1H,J_(2′,5′)=2.7 Hz, 2′-H), 7.55 (dd, 1H, J_(5′,6′)=9.0 Hz,J_(2′,6′)=2.7 Hz, 6′-H), 7.16 (s, 1H, 8-H), 6.97 (d, 1H, J_(5′,6′)=9.0Hz, 5′-H), 3.93 (s, 3H, —OCH₃), 3.91 (s, 3H, —OCH₃); IR (KBr) υ_(max):3448, 2842, 1623, 1506, 1423, 1241 cm⁻¹; GC/MS m/z: 341(M⁺, 100.00),326(M⁺ —CH₃, 98.50), 310(M⁺ —OCH_(3.) 12.5), 295(9.0.), 189(13.5),155(13.8).

Example 3 Lipid Lowering Effects of WHI-P164 in HypercholesterolemicC57B1/6 Mice.

A high cholesterol and high fat diet was used to inducehypercholesterolemia in C57B1/6 mice, using the methods described byPaigen, et al. (Nishina, et al., 1993, J. Lipid Res. 34:1413-1422) andas described for Example 1. After two weeks, these mice exhibited astable hypercholesterolemia with 4-times higher serum total cholesterollevels than control mice on regular rodent chow 451.4±14.8, n=69 versus117.8±4.8, n=22, p<0.0001). Hypercholesterolemic mice treated withWHI-P164 for one week at 1.6 mg/kg/day dose level exhibited a 23%reduction in mean serum total cholesterol levels (348.6±27.6 mg/dL, n=9versus 451.4±14.8 mg/dL, n=69, p=0.02).

As shown by their serum lipoprotein profiles (FIG. 1),hypercholesterolemic C57B1/6 mice preferentially accumulated cholesterolrich VLDL (171±19 mg/dL, n=4 versus 7±4 mg/dL, n=3, p=0.0008) but nochanges in HDL cholesterol levels (90±9 mg/dL, n=4 versus 86±13 mg/dL,n=3, p=0.8) (Table 2). WHI-P164 treatment caused a dramatic reduction ofVLDL-C (FIG. 1). In WHI-P164 treated mice, serum VLDL-C decreasedimmediately from 171±19 mg/dL (n=4), to 99±13 mg/dL (n=4) at the 1.6mg/kg/day dose level (P=0.02) and further decreased to 78±15 mg/dL (n=4)at the 16 mg/kg/day dose level (p=0.01) (Table 1). The calculatedeffective dose (ED₅₀) of WHI-P164 was 0.4 mg/kg/day with minimum serumVLDL-C level of 94±9 mg/dL. Thus, WHI-P164 was only able to reduceVLDL-C by 45%. WHI-P164 treatment did not significantly affect serumHDL-C levels (FIG. 1 and Table 1), even though there was a trend fordecreasing HDL-C with increasing WHI-P164 dose levels.

TABLE 1 Reduction of VLDL-C by WHI-P164 Dose VLDL-C HDL-C (mg/kg) N(mg/dL) p (mg/dL) p apo e^(+/+) (C57B1/6) Rodent Chow 0.0 3 7 ± 4 — 86 ±13 — Cocoa Butter Diet 0.0 4 171 ± 19  — 90 ± 9  — 1.6 4 99 ± 13 0.02 93± 8  0.8 4.0 4 106 ± 15  0.04 86 ± 5  0.7 8.0 3 101 ± 7  0.03 81 ± 9 0.5 16.0  4 78 ± 15 0.01 74 ± 10 0.3 apo e^(−/−) (C57B1/6) 0.0 4 516 ±158 — 21 ± 3  — Rodent Chow 8.0 8 190 ± 13  0.01 50 ± 10  0.09 Westerndiet 0.0 3  1296 ± 118** — 50 ± 9  — 40.0  2 587 ± 4  0.02 90 ± 4   0.04Plasma VLDL-C and HDL-C were calculated based on the lipoproteinprofiles. Treated and untreated groups were compared by student t-test.*P-values refer to differences between vehicle-treated and WHI-P164treated mice. **P = 0.01 for the difference of VLDL-C levels of apoe^(−/−) via the rodent chos vs. western diet (1296 ± 118 mg/dL vs. 516 ±158 mg/dL.

WHI-P164 treatment at the indicated dose levels, which were much lowerthan the highest nontoxic dose of 2 kg/kg, was not associated with anyobvious clinical or laboratory signs of toxicity. In particular, liverenzymes (ALT, ALP, LDH) and BUN/Creatinine remained within normal limits(Table 2). Histopathologic examination of tissues did not reveal anydrug related toxic lesions.

TABLE 2 Blood Chemistry of Treated C57B1/6 Mice 1 week 2 weeks +P164−P164 +P164 −P164 Net weight −0.26 ± 0.22 0.87 ± 0.33 1.88 ± 0.42 3.07 ±0.50 change (g) (n = 17) (n = 10) (n = 5) (n = 10) ALT — — 148 ± 54 177± 19 (IU/L) (n = 5) (n = 10) ALP 101 ± 3 110 ± 9 94 ± 10 132 ± 6 (IU/L)(n = 5) (n = 5) (n = 5) (n = 10) LD-L 1402 ± 144 1533 ± 189 459 ± 83 426± 42 (IU/L) (n = 5) (n = 5) (n = 5) (n = 9) BUN 25.4 ± 2.0 27.6 ± 2.0 —— (mg/dL) (n = 5) (n =5) Glucose 201 ± 18 144 ± 9 (mg/dL) (n = 25) (n =24)

Example 4 Lipid Lowering Effects of WHI-P164 in Hypercholesterolemic ApoE-Deficient Mice

ApoE, a component of hepatic VLDL and intestinal chylomicron, controlsthe catabolism of these particles by serving as a ligand for LDLreceptor and LDL-like receptor. Functionally defective apo e mutation isassociated with type III dyslipoproteinemia, a condition mimicked by apoe^(−/−) mice. As apo e^(−/−) mice exhibited hypercholesterolemia even inthe absence of dietary cholesterol supplementation due to a delayedclearance of hepatic VLDL particles, WHI-P164 was analyzed for itsability to reduce VLDL-C.

On regular rodent chow, the serum total cholesterol level of apo e^(−/−)mice was 836±57 mg/dL (n=47). After one week therapy with 8 mg/kg/dayWHI-P164 (n=12), the mean serum total cholesterol level was reduced to551±29 mg/dL (p=0.02). Even in the absence of dietary cholesterolchallenge, these apo e^(−/−) mice exhibited higher VLDL-C (516±158mg/dL, n=4 versus 7±4 mg/dL, n=3, p=0.04) and lower HDL-C levels thanapo e^(+/+) C57B1/6 mice on rodent chow (21±3 mg/dL, n=4 versus 86±13mg/dL, n=3, p=0.0024) (Table 1). After one week therapy with 8 mg/kg/dayWHI-P164, VLDL-C was decreased from 516±158 mg/dL, n=4, to 190±13 mg/dL,n=8, p=0.01 and HDL-C was slightly increased from 21±3 mg/dL, n=4, to50±10 mg/dL (Table 1, FIG. 2).

When challenged with a cholesterol-rich diet, these animals developedfrank hypercholesterolemia due to delayed clearance of intestinalchylomicrons which shuttle the dietary cholesterol from the intestine tothe liver. In the present study, all of the 46 ApoE-deficient mice thatwere fed Western diet (14) became severely hypercholesterolemic with amean serum total cholesterol level of 1491±59 mg/dL. One week WHI-P164therapy reduced the mean serum total cholesterol level ofhypercholesterolemic apo e^(−/−) mice to 1194±84 mg/dL (p=0.006) at adose level of 8 mg/kg/day (n=19) and to 876±57 mg/dL (p<0.0001) at adose level of 40 mg/kg/day (n=1 7) (Table 3). As shown in Table 1,dietary cholesterol challenge increased VLDL-C levels from 516±158mg/dL, (n=3) to 1296±118 mg/dL (n=3), p=0.01. After one week of therapywith 40 mg/kg/day WHI-P164, VLDL-C was decreased from 1296±118 mg/dL,n=3, to 587±4 mg/dL, n=2, p=0.02 and HDL-C was slightly increased from50±9 mg/dL, n=3, to 90±4 mg/dL, n=2, p=0.04 (Table 1, FIG. 3). Incontrast to WHI-P164, other dimethoxyquinazoline derivatives did notexhibit cholesterol-lowering activity in C57B1/6 or apo e^(−/−) mice(Table 4).

TABLE 3 Structure Function Evaluation of WHI-P164 and OtherDimethoxyquinazoline Derivatives ^(a)apo e^(−/−)(C57B1/6) ^(b)apoe^(+/+)(C57B1/6) Cholesterol Cholesterol Compounds N (mg/dL) N (mg/dL)Vehicle 46  1491 ± 59 69  451 ± 15 WHI-P97 5 1129 ± 46 10  413 ± 18WHI-P111 5 1425 ± 92 4 406 ± 11 WHI-P131 4 1532 ± 77 5 433 ± 20 WHI-P1325 1774 ± 61 — ND WHI-P154 5  1492 ± 110 — ND WHI-P164 17   876 ± 57* 9 349 ± 28** WHI-P197 5  1621 ± 146 5 372 ± 20 ^(a)apo e^(−/−)(C57B1/6)mice were placed on Western diet for two weeks, followed by treatmentwith the drug for 1 week at 40 mg/kg/day. ^(b)apo e^(+/+)(C57B1/6) micewere placed on Cocoa butter diet for two weeks, followed by treatmentwith the drug for 1 week at 1.6 mg/kg/day. *p < 0.0001 **p = 0.02.

WHI-P164 therapy, at either 8 mg/kg/day or 40 mg/kg/day, was notassociated with any obvious clinical or laboratory signs of toxicity. Inparticular, liver enzymes (ALT, AST, ALP, LDH), CPK levels, andBUN/Creatinine remained within normal limits. Histopathologicexamination of tissues did not reveal any drug related toxic lesions.

Example 5 Effects of WHI-P164 on Triglyceride (TG) Synthesis

apo e^(−/−) mice accumulate large amounts of TG-rich lipid droplets intheir livers, due a blocked VLDL-TG secretion (Kuipers et al., 1997, J.Clin. Invest. 100:2915-2922). Treatment with 40 mg/kg/day WH-P164 for 1week signicantly reduced the Oil Red O stainable lipid material in thelivers of Apo E-deficient mice that were fed on a Western diet (FIGS.4A-4D). Image analysis demonstrated a 75% reduction of lipid material intreated mice (658±256 red pixels/field, n=11 versus 2590±401 redpixels/field, n=12; p=0.0007). When tissue lipids were measured, therewas a 22% reduction in hepatic cholesterol 39% reduction in hepatictriglycerides. As shown in Table 4, the reduction in hepatic TGaccumulation was not due to depletion of precursors for hepatic TGaccumulation, namely plasma free fatty acid (FFA) and triglyceride.

TABLE 4 Analysis of Tissue Lipids Control Lipid Levels WHI-P164(vehicle) p Value hepatic cholesterol 51.4 ± 4.4 65.9 ± 5 p = 0.5 (mg/gwet wt) (n = 7)  (n = 8)  hepatic triglycerides 211.6 ± 27   349.3 ± 34p = 0.008 (mg/g wet wt) (n = 7)  (n = 8)  plasma FFA  3.6 ± 0.3   3.2 ±0.3 (mM) (n = 12) (n = 11) plasma TG 165 ± 19   167 ± 14 (mg/dL) (n =15) (n = 17)

Example 6 Regression of Pre-existing Atherosclerotic Lesions

The effectiveness of WHI-P164 against pre-existing atheroscleroticlesions in apo e^(−/−) mice kept 7 months on high fat Western diet wasexamined. apo e^(−/−) mice were treated with either vehicle control orWHI-P164 at 40 mg/kg/day for one month. As shown in FIG. 6A, beforetreatment, fatty streaks stainable by Sudan IV covered 40±5% (n=3) ofthe aortic surface of these mice. Treatment with WHI-P164 caused amarked regression of the atherosclerotic lesions with fatty streakscovering only 23±2% (n=7) of the aortic surface (FIG. 6B), as comparedto 40±5% in untreated mice (n=3) (FIG. 6A) (p=0.002) and 40±5% in thevehicle-treated mice (n=8) (FIG. 6C) (p=0.007).

Example 7 Effect of WHI-P164 on Liver Lipoprotein SynthesisDetermination of In Vivo Hepatic VLDL-TG Production Using Triton WR1339

To determine if WHI-P164 prevent hepatic TG accumulation by relieving 9block in VLDL-TG secretion or by inhibiting upstream TG synthesis, invivo hepatic VLDL-TG synthesis was analyzed. Synthesis was determined byfollowing the accumulation of VLDL-TG after shutting down VLDLcatabolism with Triton WR1339 injection and intestinal chylomicronsynthesis with a 36 hours fast.

C57B1/6 and apoE-deficient mice were treated with intra peritonealinjections of vehicle or 40 mg/kg WHI-P164 (in 50 μl vehicle) for 7consecutive days, followed by a 36 hour fast to shut down intestinalchylomicron synthesis. Subsequently, mice were injected intraperitonealy(i.p.) with 500 mg/kg Triton WR1339 (Sigma Chemical Co.) in 0.9% NaCl tocompletely inhibit their plasma VLDL clearance, as previously reported(Kuipers et al., 1996, Heptology 24:241-247). Blood samples (50 μl) weretaken at 0 and 4 hours after Triton WR1339 injection and plasma TG wasmeasured enzymatically using a commercially available enzymatic kit(Sigma Chemical Co.), as previously reported (Kuipers, supra).

WHI-P164 treated apo e^(−/−) mice exhibited significantly lessaccumulation of VLDL-TG than vehicle-treated control mice after TritonWR1339 treatment (p=0.0002), although they exhibited similar pre-TritonWR1339 serum triglyceride levels (Table 5). WHI-P164 treated C57B1/6mice also exhibited significantly less accumulation of VLDL-TG thanvehicle-treated control mice (p=0.003), although they exhibited similarpre-Triton WR1339 serum triglyceride levels (Table 5). The data supportthe hypothesis that WMI-P164 inhibited TG synthesis, thereby preventinghepatic accumulation of TG in apo e^(−/−) mice.

TABLE 5 Inhibition of Hepatic VLDL-TG Synthesis by WHI-P164 Triglyceride(mg/dL) Pre-Triton WR 1339 Post-Triton WR 1339 apo e^(−/−)(C57B1/6)Vehicle 110 ± 12 (n = 8)  744 ± 37 (n = 14) WHI-P164 155 ± 14 (n = 10) 465 ± 46 (n = 8)* apo e^(+/+)(C57B1/6) Vehicle 143 ± 6 (n = 5) 1354 ±44 (n = 12) WHI-P164 124 ± 12 (n = 5) 1031 ± 95 (n = 8)** HepaticVLDL-TG was determined as accumulation of triglyceride after injectionof Triton WR1339, which shuts down VLDL catabolism, and 36 hours fastwhich shuts down intestinal chylomicron synthesis. Student t-test wasused to compare the WHI-164 treated group versus the vehicle treatedgroup. *p = 0.0002 **p = 0.003

The decrease in TG synthesis was not specific to the liver, asintestinal chylomicron-TG was also inhibited by WHI-P164 therapy. When36 hours fasted mice were fed again, they immediately consumed the foodand exhibited a time dependent accumulation in plasma TG, due tointestinal chylomicron-TG synthesis. Postprandial accumulation wasinhibited by WHI-P164 therapy (FIG. 5). Inhibition reached statisticalsignificance at 6 hours in vehicle treated mice having a plasma TG levelof 272±25 mg/dL, n=5, as compared to a plasma TG level of 155±32 mg/dL,n=5, p=0.004 in WHI-P164 treated mice.

The present invention should not be considered limited to the particularexamples described above, but rather should be understood to cover allaspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable will bereadily apparent to those of skill in the art to which the presentinvention is directed upon review of the instant specification.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated by reference.

We claim:
 1. A pharmaceutical composition comprising: an effectivecholesterol lowering amount of a combination comprising: a compound ofthe formula:

wherein X comprises a straight or branched chain or cyclic alkyl, or anaromatic ring structure, optionally substituted with R, wherein R ishalogen, hydroxy or lower alkyl, and wherein each of R₁-R₅ areindependently selected from H, OH, NH₂, SH, alkyl, alkoxy, and acyloxy;an inhibitor of HMG CoA reductase; and a pharmaceutically acceptablevehicle.
 2. The composition according to claim 1, wherein the compoundhas the formula:

wherein each of R₃-R₄ independently comprise H, OH, alkoxy, acyloxy, SH,NH₂ or halo, and R is halogen, hydroxyl and lower alkyl.
 3. Thecomposition according to claim 1, wherein the compound is4-(3′-bromobenzoyl)-6,7-dimethoxy quinazoline.
 4. A method of reducingblood cholesterol in a subject comprising administering to the subjectan effective cholersterol lowering amount of a combination comprising: acompound of the formula:

wherein X comprises a straight or branched chain or cyclic alkyl, or anaromatic ring structure, optionally substituted with R, wherein R ishalogen, hydroxyl, and lower alkyl, and wherein each of R₁-R₅ areindependently selected from H, OH, NH₂, SH, alkyl, alkoxy, and acyloxy;an inhibitor of HMG CoA reductase; and a pharmaceutically acceptablevehicle.
 5. The method according to claim 4, wherein the compound hasthe formula:

wherein each of R₃-R₄ independently comprise H, OH, alkoxy, acyloxy, SH,NH₂ or halo, and R is halogen, hydroxyl and lower alkyl.
 6. The methodaccording to claim 4, wherein the compound is4-(3′-bromobenzoyl)-6,7-dimethoxy quinazoline.
 7. The method accordingto claim 4, wherein the compound is admistered at a daily dose of fromabout 50 to about 500 mg/day.